A controlled-release diabetic wound healing treatment method

Abstract

The present invention relates to the technical field of diabetes, and in particular to a controlled-release diabetic wound healing promoting treatment method, comprising the following treatment experimental steps: step S1: preparation of Hst1@PNPs; step S2: Coomassie brilliant blue staining; step S3: scanning and transmission electron microscopy detection; in the present invention, a slow-release Hst1 treatment method is constructed by encapsulating Hst1 into platelet membrane vesicles derived from autologous blood. On the one hand, the PNPs encapsulate the linear Hst1 peptide structure to prevent it from being rapidly degraded by high proteases in diabetic wound exudate, thereby improving bioavailability. On the other hand, the PNPs can adsorb excess LPS in the wound exudate, and cooperate with Hst1 to achieve anti-inflammatory and anti-oxidative stress effects. Subsequent cell and animal experiments further confirmed that Hst1@PNPs have the effects of anti-inflammatory, scavenging ROS, promoting re-epithelialization, collagen deposition and angiogenesis, thereby promoting diabetic wound healing.

Description

Technical Field

[0001] The invention relates to the technical field of diabetes, in particular to a controlled-release diabetic wound healing treatment method. Background Art

[0002] Diabetes is a syndrome of metabolic disorders of sugar, protein, fat, water and electrolytes caused by multiple pathogenic factors acting on the body, leading to pancreatic islet dysfunction, insulin resistance, etc. It is characterized by persistent pathological hyperglycemia. The biggest threat to diabetic patients comes from complications such as retinopathy, diabetic nephropathy, neuropathy and difficult wound healing. Its treatment is an especially important part of diabetes treatment.

[0003] As the largest organ in the human body, the skin will inevitably be affected by the metabolic disorders of diabetes and suffer corresponding multiple injuries. The difficulty in healing skin wounds caused by exogenous trauma of diabetes has become one of the most common serious complications in diabetic patients. It is characterized by a long course of disease, complex and easy recurrence, high incidence, difficult treatment, limited treatment methods, poor prognosis, and this complication is often difficult to cure. Therefore, based on the above problems, a controlled release diabetic wound healing treatment method is proposed. Summary of the invention

[0004] The purpose of the present invention is to provide a controlled-release diabetic wound healing treatment method to solve the problem that the existing diabetic complication skin wounds cannot be healed for a long time.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A controlled-release diabetic wound healing treatment method comprises the following treatment experimental steps:

[0007] Step S1: Preparation of Hst1-AIE@PNPs;

[0008] Step S2: Coomassie Brilliant Blue staining;

[0009] Step S3: Characterization of Hst1-AIE@PNPs;

[0010] Step S4: in vitro sustained release of Hst1-AIE@PNPs;

[0011] Step S5: in vitro LPS adsorption;

[0012] Step S6: cell culture;

[0013] Step S7: CCK-8 cytotoxicity assay;

[0014] Step S8: cell scratch test;

[0015] Step S9: DCFH-DA experiment;

[0016] Step S10: in vitro immunofluorescence experiment;

[0017] Step S11: in vitro enzyme-linked immunosorbent assay;

[0018] Step S12: high-throughput sequencing;

[0019] Step S13: animal experiment;

[0020] Step S14: diabetic mouse wound healing experiment;

[0021] Step S15: histological staining;

[0022] Step S17: In vivo ELISA.

[0023] Preferably, the following treatment experiment effect verification steps are included:

[0024] Step S1: Hst1@PNPs characterization;

[0025] Step S2: In vitro experiments to verify the effect of Hst1@PNPs on the migration of HaCaT cells and fibroblasts;

[0026] Step S3: in vitro experiments to verify the ROS scavenging ability of Hst1@PNPs;

[0027] Step S4: in vitro experiments to verify the effect of Hst1@PNPs on macrophage polarization and expression of pro-inflammatory factors in vitro;

[0028] Step S5: verify the sustained release effect of Hst1@PNPs in vivo and its effect on promoting wound healing in diabetic mice skin;

[0029] Step S6: In vivo experiments to verify the effects of Hst1@PNPs on wound re-epithelialization, collagen expression and angiogenesis;

[0030] Step S7: In vivo experiments verify the regulatory effects of Hst1@PNPs on macrophage polarization, inflammatory factors, and oxidative stress factor expression;

[0031] Step S8: High-throughput sequencing was used to detect the regulatory effect of Hst1 on RAW264.7 macrophages.

[0032] Compared with the prior art, the present invention has the following beneficial effects:

[0033] In the present invention, a sustained-release Hst1 treatment method is constructed by encapsulating Hst1 into platelet membrane vesicles derived from autologous blood. On the one hand, the PNPs-encapsulated linear Hst1 peptide structure can prevent it from being rapidly degraded by high proteases in diabetic wound exudate, thereby improving bioavailability. On the other hand, PNPs can adsorb excess LPS in wound exudate and synergize with Hst1 to achieve anti-inflammatory and anti-oxidative stress effects. Subsequent cell and animal experiments further confirmed that Hst1@PNPs has anti-inflammatory, ROS-scavenging, re-epithelialization, collagen deposition and angiogenesis promoting effects, thereby promoting diabetic wound healing and solving the problem of long-term treatment of existing diabetic complications of skin wounds. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 It is an overall schematic diagram of the present invention;

[0035] Figure 2 Schematic diagram used for characterization of Hst1@PNPs of the present invention;

[0036] Figure 3 Schematic diagram of the effect of Hst1@PNPs on the migration of HaCaT cells and fibroblasts and the ability of Hst1@PNPs to scavenge ROS verified by in vitro experiments of the present invention;

[0037] Figure 4 Schematic diagram of the in vitro experiment used to verify the effect of Hst1@PNPs on macrophage polarization and in vitro pro-inflammatory factor expression in the present invention;

[0038] Figure 5 This is a schematic diagram used in the present invention to verify the sustained release effect of Hst1@PNPs in vivo and its effect on promoting the healing of skin wounds in diabetic mice;

[0039] Figure 6 Schematic diagram of the in vivo experiment used to verify the effect of Hst1@PNPs on wound re-epithelialization, collagen expression and angiogenesis in the present invention;

[0040] Figure 7 Schematic diagram of the in vivo experiment used to verify the regulatory effect of Hst1@PNPs on macrophage polarization, inflammatory factors and oxidative stress factor expression in the present invention;

[0041] Figure 8 This is a schematic diagram of the high-throughput sequencing used in the present invention to detect the regulatory effect of Hst1 on RAW264.7 macrophages; Figure 8-1 yes Figure 8 Figure A; Figure 8-2 yes Figure 8 Figure B; Figure 8-3 yes Figure 8 Figure C; Figure 8-4 yes Figure 8 D figure; Figure 8-5 yes Figure 8 Figure E; Figure 8-6 yes Figure 8 Figure F. DETAILED DESCRIPTION

[0042] See also Figure 1-8 , the present invention provides a technical solution:

[0043] A controlled-release diabetic wound healing treatment method comprises the following treatment experimental steps:

[0044] Step S1: Preparation of Hst1@PNPs,

[0045] 50 ml of whole blood was drawn from the volunteers. The collected blood was centrifuged for the first time at 600 g for 10 minutes to remove the red blood cells at the bottom layer. The second centrifugation was performed at 3000 rpm for 20 minutes to remove the upper serum layer. The platelet precipitate was washed with 1 ml of The platelets were suspended in PBS to obtain a platelet concentration of 1249×10^9 / L. The concentrated platelets were repeatedly frozen and thawed three times at -80℃ and 25℃, and then centrifuged at 12000g for 10 minutes at 4℃ to collect the platelet membrane (PM) precipitate. The platelet membrane was fully ground with a grinding rod and then centrifuged at 3200g for 5 minutes. The precipitate was suspended in PBS, and the suspension was extracted with a syringe and installed in an engineered vesicle extrusion device. It was repeatedly passed through a 0.8um membrane and a 0.2um membrane for 20 times to obtain PNPs and stored at -80℃. The concentrations of PM and PNPs were detected using a BCA kit (P0012S, Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China), which were 7.16g / L and 4.37g / L, respectively. PNPs were mixed with Hst1 and shaken at 4℃ for 12 hours to obtain Hst1@PNPs.

[0046] Step S2: Coomassie Brilliant Blue staining,

[0047] 10% separation gel and stacking gel were prepared. After the gel was formed, the glass plate was placed in the electrophoresis tank, 1× electrophoresis solution was added, protein markers, PM, and PNPs were added to the sample wells, and the gel was run at 80 V for 20 min, then at 120 V for 60 min. The gel was cut and soaked in distilled water for 5 min, and then Coomassie Brilliant Blue staining solution (G1042, Beijing Labread Trading Co., Ltd., Beijing, China) was added, incubated on a shaker for 1 h, destained with tap water for 30 min, and then photographed with a chemiluminescence imaging system;

[0048] Step S3: Characterization of Hst1@PNPs,

[0049] The Hst1, PNPs, and Hst1@PNPs samples were dehydrated and frozen under liquid nitrogen, then cut into thin slices with a diamond blade, a layer of conductive material was coated on the samples using a vacuum sputtering instrument, and then placed in a SEM (Nova Nano 450, Thermo Fisher Scientific, USA) and TEM (Talos F200, Thermo Fisher Scientific, USA) chamber, and the surface and internal structure and morphology of the samples were detected by scanning and transmission electron microscopy, and the PNPs, Hst1, and Hst1@PNPs samples were diluted with ultrapure water to a particle concentration suitable for dynamic light scattering (DLS) analysis, and the particle size of these samples was detected using an Anton Paar Litesizer 100 device and a fluorescence spectrometer (NanoDrop 3300, Thermo Fisher Scientific, USA);

[0050] Step S4: In vitro sustained release of Hst1@PNPs,

[0051] 1 ml of Hst1@PNPs was added to a dialysis bag with a pore size of 8000 kDa, which was then placed in a 15 ml EP tube filled with 5 ml of distilled water. After 0.5 h, 1.5 h, 3 h, 6 h, 8 h, 12 h and 24 h, 100 ul of solution was taken from the 5 ml of distilled water, and these samples were added to a 96-well plate, and the OD value was detected at a wavelength of 450 nm using an enzyme reader. The exudate from the diabetic wound of the rat was collected, and then the collected exudate was mixed with Hst1 and Hst1@PNPs in a volume ratio of 1:1, and 1 ml of the mixture was added to the dialysis bag. The subsequent steps were the same as above. The OD values ​​of these samples were detected after 0.5 h, 1 h, 3 h, 6 h, 8 h, 12 h and 24 h, and then graphed using Graphpad prism9.

[0052] Step S5: In vitro adsorption of LPS,

[0053] 10 ng / ml LPS was mixed with 10 ug / ml, 100 ug / ml, and 500 ug / ml of PNPs, respectively, and then fully shaken on a shaker at 37 °C for 1 h. The mixture was centrifuged at 4500 rpm for 6 min, and the supernatant was collected. The transmittance of the supernatant was measured using a Fourier transform infrared spectrometer (Nicolet iS50 FTIR, Thermo Fisher Scientific, USA), and the LPS concentration in the supernatant was measured using a horseshoe crab kit (EC32545, Xiamen Boendu Biotechnology Co., Ltd., Xiamen, China) according to the instructions;

[0054] Step S6: Cell culture,

[0055] RAW 264.7 macrophages, HaCaT cells, and human skin fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics, and the cells were cultured in an incubator at 37°C and 5% CO2;

[0056] Step S7: CCK-8 cytotoxicity assay,

[0057] RAW 264.7 cells were seeded in 96-well plates at a density of 2×10^4 cells per well and incubated overnight. In previous studies, 10 μM Hst1 was verified to be the optimal concentration for promoting skin cell migration, adhesion and acute wound healing. Therefore, 10 μM Hst1 was used as a positive control in this study. The cells were treated with 10 μM Hst1, 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml PNPs, Hst1@0.1 μg / ml PNPs, Hst1@0.5 μg / ml PNPs, and Hst1@1 μg / ml PNPs for 48 hours, respectively. At 24 hours and 48 hours after treatment, 10 μl of CCK-8 solution was added to each well and incubated for another hour. Subsequently, the absorbance at a wavelength of 450 nm was measured using a microplate reader. According to the experimental results, the optimal concentration of 1 μg / ml PNPs was used in subsequent experiments.

[0058] Step S8: Cell scratch test,

[0059] HaCaT cells and human skin fibroblasts were seeded into 6-well plates until 100% confluence was achieved. Two straight lines were drawn in each well using a sterile 200 μl pipette tip. After rinsing with PBS, the cells were exposed to the control group (DMEM+1% FBS), the experimental groups 10 μM Hst1+DMEM+1% FBS, 1 μg / ml PNPs+DMEM+1% FBS and Hst1@1 μg / ml PNPs+DMEM+1% FBS, respectively. Immediate images after scratching, as well as images at 24 h, 48 h, 72 h and 96 h after scratching were taken using a Nikon optical microscope. The unclosed area of ​​the scratch wound was measured using Image J software, and the percentage of wound healing was calculated using the formula: percentage of wound healing (%) = [(A0-Ax) / A0] × 100%, where A0 represents the initial wound area, and Ax represents the wound area at 24 h, 48 h, 72 h and 96 h;

[0060] Step S9: DCFH-DA experiment,

[0061] RAW264.7 macrophages were seeded in 24-well plates at a density of 1×10^5 cells per well and grown to 60%. The cells were treated with Hst1, PNPs, and Hst1@PNPs for 8 h, respectively. Then, the medium was removed, and 100 ng / ml of LPS was added to each well (except the control group) to induce ROS generation and incubated for 24 h. Subsequently, 100 μl of serum-free medium containing 0.1 μl DCFH-DA (S0033S, Shanghai Bio-Technology Co., Ltd., Shanghai, CN) was added to each well and incubated at 37 °C in the dark for 50 min. 0.1 μl of Hochest was added to each well to stain the cell nucleus for 10 min, and then rinsed with PBS three times and photographed with a fluorescence confocal microscope (Nikon Eclipse TI-SR, Tokyo, Japan);

[0062] Step S10: In vitro immunofluorescence assay,

[0063] RAW264.7 cells were seeded in 24-well plates at a density of 1×10^5 cells per well and incubated overnight. Subsequently, the cells were treated with control, LPS, LPS+Hst1, LPS+PNPs, and LPS+Hst1@PNPs for 24 hours, respectively. After rinsing with PBS three times, the cells were fixed in 4% paraformaldehyde for 20 minutes, rinsed with PBS again for three times, and 0.5% Triton-X-100 was added to the plate. After incubation at room temperature for 10 minutes, the cells were blocked with 5% bovine serum albumin (BSA) for 30 minutes, and then the primary antibodies iNOS (53-5920-80; 1:100; Thermo Fisher Scientific Co., Ltd, California, US) and CD206 (ab300621; 1:50; Abcam Trade Co., Ltd, Shanghai, CN) at 37°C in the dark for 2 h, and then incubated with the corresponding fluorescent secondary antibody for another 1 h at room temperature. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and photographed using a fluorescent confocal microscope. In order to distinguish between M1 and M2 macrophages, iNOS positive staining was used to characterize M1 macrophages, and CD206 positive staining was used to characterize M2 macrophages;

[0064] Step S11: In vitro ELISA,

[0065] After the cells were treated under different conditions, the culture supernatant of each well was collected, and the expression levels of IL-6 (RE3262-96T; Bioroyee Biotech Co., Ltd, Beijing, CN), IL-1β (WKQ-SJH-01413-96T; Wekeqi Biotech Co., Ltd, Chengdu, CN), and TNF-α (1217202; Dakewei Biotech Co., Ltd, Shenzhen, CN) were determined using ELISA kits, and the absorbance was measured at the corresponding wavelength using a microplate reader, and the concentrations of these cytokines were calculated using the standard curve formula;

[0066] Step S12: high-throughput sequencing,

[0067] RAW264.7 macrophages were seeded in 12-well plates and then exposed to the control group, LPS group, and LPS+Hst1 group for 24 hours to induce macrophage polarization. Then, total RNA was extracted using Trizol reagent, 1 μg of total RNA was used for subsequent library preparation, poly(A) mRNA was isolated using Oligo(dT) beads, mRNA fragmentation was performed using divalent cations and high temperature, primer synthesis was performed using random primers, first-strand cDNA and second-strand cDNA were synthesized, and the purified double-stranded cDNA was subjected to end repair and A-tailing reaction, and adapters were added to both ends by TA ligation. Then, PCR amplification was performed for each sample using P5 and P7 primers, and the PCR products were verified. Next, the libraries with different indexes were multiplexed and loaded onto IlluminaHiSeq / Illumina Novaseq / MGI2000 instruments, and 2x150 paired-end (PE) configuration sequencing was performed according to the instructions. The final library was quantified by a fluorometer and analyzed using Bioanalyzer The 2100 system checked the size distribution to ensure that it met the specific requirements of the sequencing platform, and then GO and KEGG analyses were performed on the differentially expressed genes;

[0068] Step S13: Animal experiment,

[0069] Thirty male C57 mice aged 6–8 weeks and weighing 22–28 g were purchased from Guangdong Animal Research Center, China. All animal experiments were approved by the Animal Ethics Committee of the Dermatology Hospital of Southern Medical University. Before the experiments, the mice were allowed to adapt to the living environment for one week;

[0070] Step S14: Diabetic mouse wound healing experiment,

[0071] Thirty C57 mice were prepared and fed with a standard diet for 3 days, then changed to a 60% high-fat diet for one week. The mice were fasted overnight, then marked with ear tags and fasting blood glucose levels were measured. Subsequently, a 10 mg / mL STZ solution was prepared using pH 4.5 sodium citrate, and STZ was then injected intraperitoneally at 100 mg / kg. STZ injections were performed on days 1, 3, and 5, respectively. Blood glucose levels were monitored every day after the three injections. If blood glucose returned to normal levels, additional injections were performed. Once blood glucose levels were stable for a week (>16.7 mmol / L), the mice could be selected for the diabetic wound model. Finally, 18 mice were successfully modeled.

[0072] The treatments were divided into four groups: control group (0.9% NaCl), Hst1 group, PNPs group and Hst1@PNPs group. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (5 ml / kg). The hair on the back of each mouse was carefully shaved and disinfected with iodine. Four acute full-thickness skin defect models were made in the back area of ​​each mouse using a 0.6 cm diameter biopsy device. Then, silicone rings were glued to the wound surface to prevent wound contracture. The four treatments were randomly applied to four wounds of each mouse for a self-control study. Then, 0.5 ml of the drug was given to each wound every other day until the wound was completely healed. The wounds of the mice were photographed on days 0, 3, 5, 7, 10 and 14 after surgery, and then the images were taken using ImageJ. The wound area of ​​mice was measured by J software, and the wound healing percentage was calculated, wound healing percentage (%) = [(A0-Ax) / A0] × 100%, A0 represents the initial wound area on day 0, Ax represents the wound area on days 3, 5, 7, 10 and 14, 5 mice were sacrificed at each time point on days 3, 7 and 14 after surgery, and the wound specimens were completely removed. The samples were divided into two parts, one part was fixed with 4% paraformaldehyde solution for subsequent histological staining, and the other part was used for Western blotting and ELISA detection;

[0073] Step S15: Histological staining,

[0074] Hematoxylin-eosin staining; Masson's trichrome staining: tissue sections were baked at 70°C for 1 hour, then dewaxed and rinsed with running water for 2 minutes, stained with iron hematoxylin for 4 minutes, then rinsed with running water, differentiated with 1% hydrochloric acid alcohol for 1 second, then rinsed with running water for 2 minutes to make it blue, tissue sections were stained with picric acid for 3 minutes, phosphomolybdic acid for 4 minutes, and aniline blue for 1 minute, then quickly rinsed and baked in an oven for 30 minutes, images were taken using an electron microscope and analyzed using Image-Pro Plus software, 5 tissue sections were selected from each group for photography, and the expression level of new collagen was quantified using the following formula: average optical density value (AOD) = total optical density (IOD) value / area sum; in vivo immunofluorescence staining: sections were repaired with ethylenediaminetetraacetic acid (EDTA) solution with pH = 9.0 for 15 minutes, blocked with 5% bovine serum albumin (BSA) at 37°C for 30 minutes, and incubated with primary antibody CD31 (AF3628, 1:200, Bio-Techne, Minneapolis, Minnesota, USA), iNOS (53-5920-80, 1:100, Thermo Fisher Scientific Inc., California, USA), CD206 (ab300621, 1:50, Abcam Trading Co., Ltd., Shanghai, China), the corresponding fluorescent secondary antibodies were added, incubated for 1 h at room temperature, and then blocked with 4-6-diamidino-2-phenylindole (DAPI) anti-fluorescence quencher, and 5 tissue sections in each group were photographed using a fluorescence confocal microscope using Image-Pro Plus software was used to measure the fluorescence area of ​​CD31, and the number of M1 and M2 macrophages was automatically counted and analyzed using Image-Pro Plus software; Western blot, in order to quantify the expression of M1 and M2 macrophages, wound tissue samples were taken for Western blot detection. First, the tissue was homogenized in lysis buffer using a homogenizer, and then the homogenate was centrifuged at 12000g for 10 minutes to obtain the supernatant. The protein concentration was determined using a BCA kit, and the sample was mixed with the loading buffer, boiled and added to the SDS-PAGE gel wells. 80 volts were selected for electrophoresis for 30 minutes, and then switched to 120 volts for electrophoresis for 1 hour. The gel was then transferred to a PVDF membrane, the membrane was blocked with skim milk powder, and then incubated with primary antibodies iNOS (ab283655, 1:1000, Abcam Trading Co., Ltd., Shanghai, China) and CD206 (ab64693, 1:1000, Abcam Trading Co., Ltd., Shanghai, China) at 4°C overnight. Subsequently, the secondary antibody was incubated at room temperature for 1 hour and controlled release was performed using chemiluminescence. Image J software to analyze the Western blot bands to detect the expression levels;

[0075] Step S16: in vivo ELISA,

[0076] To quantify the expression of inflammatory and oxidative stress factors, wound tissue samples were homogenized in lysis buffer using a homogenizer and then centrifuged at 12,000 g for 10 min to obtain the supernatant, and the levels of IL-6, IL-1β, IL-10, TNF-α, ROS (JL20383; Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China), malondialdehyde (MDA) (MM-0897M1; Guangzhou Anfei Biotechnology Co., Ltd., Guangzhou, China), and superoxide dismutase 2 (SOD2) (MM-0389M1; Guangzhou Anfei Biotechnology Co., Ltd., Guangzhou, China) were measured using ELISA kits, and the subsequent steps were the same as the above cell ELISA experiments;

[0077] Step S17: Statistical analysis,

[0078] Statistical analysis was performed using one-way ANOVA or two-way ANOVA followed by Bonferroni test. All numerical results were expressed as mean ± standard deviation (SD), and a significance level of P < 0.05 was considered statistically significant.

[0079] The treatment experiment effect verification steps include the following:

[0080] Step S1: Hst1@PNPs characterization,

[0081] First, PNPs were synthesized and Coomassie Brilliant Blue staining confirmed that PNPs retained most of the protein distribution in PM. Electron microscopy revealed that Hst1 presented a square structure and could be encapsulated by PNPs with a ring-shaped lipid membrane structure to form a nanoparticle structure. DLS analysis showed that the particle sizes of PNPs, Hst1, and Hst1@PNPs were approximately 174.1 nm, 636.1 nm, and 1216.0 nm, respectively. The particle size of PNPs increased by hundreds of times after encapsulating Hst1, indicating that Hst1 was not only encapsulated in PNPs, but also likely bound to the surface membrane proteins of PNPs.

[0082] Studies have shown that Hst1 is degraded to 46% and 8% after incubation with chronic wound exudate for 8 hours and 24 hours, respectively. The half-life of platelets in systemic circulation is about 30 hours, which is partly attributed to the interaction between CD47 on them and signal regulatory protein α (SIRPα) on immune cells to prevent them from being cleared by the immune system. PNPs retain these characteristics of platelets and can achieve sustained release and targeted delivery of drugs when encapsulated. In this study, we encapsulated Hst1 in PNPs to achieve sustained release, and verified it through in vitro release experiments. Through dialysis, Hst1 can cross to the external solution through a dialysis bag with a certain pore size, and then collect the solution to detect OD The results showed that Hst1@PNPs can release Hst1 slowly over time and reach a plateau at 8 hours. Subsequently, in the diabetic wound exudate of rats, Hst1 was degraded to 33.6% and 50.4% of its original amount after 0.5 hours and 24 hours of co-incubation, respectively. Obviously, Hst1@PNPs was more stable, with 90.1% and 76.1% remaining after 0.5 hours and 24 hours. Therefore, compared with the previous study that only 8% of Hst1 was degraded after 24 hours, Hst1@PNPs can increase the Hst1 retention rate by 9.5 times in the high protease environment of diabetic wound exudate, greatly improving the treatment efficiency and reducing the frequency of administration.

[0083] LPS can promote macrophage polarization to M1 phenotype, which can initiate pro-inflammatory response and secrete pro-inflammatory factors such as IL-6, IL-1β and TNF-α. In this study, three different concentrations of PNPs (10ug / ml, 100ug / ml, 500ug / ml) were used to adsorb 10ng / mL LPS, and the transmittance of the solution was detected by infrared spectrometer. It was found that the transmittance of PNPs with a concentration of 100ug / ml was much higher than that of PNPs with other concentrations, indicating that the adsorption effect of LPS was more obvious. In addition, the use of chromogenic matrix horseshoe crab kit confirmed that PNPs with three concentrations can significantly adsorb LPS. Therefore, PNPs can adsorb excess LPS in diabetic wound exudate to improve the microenvironment of persistent inflammation and high-intensity oxidative stress, and synergize with Hst1 to promote wound healing.

[0084] Previous studies have confirmed that Hst1 can promote the adhesion, crawling and migration of various cells. In the concentration range of 0.5uM to 400uM, LDH experiments confirmed that no obvious cytotoxicity was found at any concentration. In addition, PNPs are derived from autologous platelets, have high biosafety and low immunogenicity, and no toxic reagents are added during the preparation process. In this study, the CCK8 kit was used to detect the cytotoxicity of three different concentrations of Hst1@PNPs, and it was found that there was no obvious cytotoxic effect on R264.7 macrophages after 24 hours and 48 hours of treatment;

[0085] Step S2: In vitro experiments to verify the effect of Hst1@PNPs on the migration of HaCaT cells and fibroblasts.

[0086] In order to study the effect of Hst1@PNPs on skin cell migration, scratch tests were performed on HaCaT cells and human skin fibroblasts. The experiments were divided into control group, 10μM Hst1, PNPs and Hst1@PNPs groups. The results showed that compared with other groups, the scratch width of HaCaT cells and fibroblasts treated with Hst1@PNPs was significantly reduced. Similarly, the wound area of ​​HaCaT cells treated with Hst1@PNPs was significantly reduced, and more than 80% healed after 48 hours. However, the control group and 10μM Hst1 positive control group had no significant promoting effect on the migration of HaCaT cells and fibroblasts (P<0.05, Figure 3 DE), the above results showed that Hst1 and PNPs had a synergistic effect on cell migration;

[0087] Step S3: In vitro experiments to verify the ability of Hst1@PNPs to scavenge ROS.

[0088] To verify the effect of Hst1@PNPs on ROS scavenging, the ROS level was evaluated by immunofluorescence. The results showed that the intracellular ROS production of Raw264.7 macrophages induced by LPS increased significantly in the LPS group, while the ROS level was significantly reduced in the LPS+Hst1, LPS+PNPs, and LPS+Hst1@PNPs groups and restored the ROS level to the level of the control group. Therefore, Hst1 and PNPs can not only clear LPS-induced ROS production alone, but also achieve a synergistic effect in ROS scavenging.

[0089] Step S4: In vitro experiments verify the effect of Hst1@PNPs on macrophage polarization and expression of pro-inflammatory factors in vitro.

[0090] In order to study the effect of Hst1@PNPs on macrophage polarization, the expression levels of M1 and M2 macrophages were detected by immunofluorescence. Compared with the control group, LPS significantly increased the expression of iNOS, a marker of M1 macrophages, and successfully induced polarization to M1 phenotype. Compared with LPS and control groups, Hst1, PNPs and Hst1@PNPs groups significantly reduced the expression of iNOS and increased the expression of CD206, a marker of M2 macrophages. Therefore, Hst1@PNPs can promote the transformation of M1 to M2 macrophages and M0 to M2, thereby promoting the transition of diabetic wound healing from the inflammatory phase to the granulation phase and remodeling phase.

[0091] In addition, to evaluate the effect of Hst1@PNPs on the production of inflammatory factors in LPS-induced macrophages, the expression levels of IL-1β, IL-6, and TNF-α were detected using Elisa kits. Compared with the control group, LPS significantly increased the expression of pro-inflammatory factors, while Hst1@PNPs could significantly reduce the expression of IL-1β and TNF-α compared with the LPS group. In addition, the IL-6 levels in the PNPs and Hst1@PNPs groups were significantly higher than those in the LPS and control groups.

[0092] Step S5: Verify the sustained release effect of Hst1@PNPs in vivo and its effect on promoting wound healing in diabetic mice skin.

[0093] A diabetic wound model was constructed to evaluate the sustained-release effect of Hst1@PNPs on diabetic wounds. Mice with blood glucose levels exceeding 16.7mmol / L for one week were identified as being useful for diabetic wound model construction. On the 3rd, 5th, 7th, 10th, and 14th days after surgery, the healing rate of diabetic wounds treated with Hst1@PNPs was faster than that of other groups. Quantitative analysis further confirmed that Hst1@PNPs significantly promoted diabetic wound healing on the 3rd and 5th days after surgery, with wound healing percentages reaching 6.85 times and 2.13 times that of the control group on the 3rd and 5th days after surgery, respectively. However, there was no statistical difference between the Hst1 group and the PNPs group and the control group. These results indicate that when Hst1 is applied to diabetic wounds, it is rapidly degraded by high proteases, while Hst1@PNPs significantly promotes diabetic wound healing through its sustained-release effect.

[0094] Step S6: In vivo experiments verify the effects of Hst1@PNPs on wound re-epithelialization, collagen expression and angiogenesis.

[0095] In this study, the effects of Hst1@PNPs on epithelial migration, collagen deposition, and angiogenesis were evaluated by pathological tissue staining. As time went on, the epidermis in the wound gradually migrated toward the center of the wound until it closed. Quantitative analysis showed that on the 3rd and 7th days after surgery, the length of the unclosed wound in the control group was significantly longer than that in the other groups. Hst1@PNPs showed the fastest epidermal migration speed among the four groups. On the 7th and 14th days after surgery, the collagen expression level in the Hst1@PNPs group was significantly higher than that in the control group. In order to study the effect of Hst1@PNPs on angiogenesis, the expression of CD31-positive blood vessels was detected by immunofluorescence staining. On the 7th day after surgery, the CD31-positive blood vessel fluorescence area in the Hst1@PNPs group and the PNPs group was significantly higher than that in the control group. However, there was no statistical difference in the Hst1 group in promoting the expression of CD31-positive blood vessels. On the 14th day after surgery, there was no statistical difference between the groups. Overall, these results showed that compared with Hst1 alone, Hst1@PNPs significantly promoted the structural integrity and functional regeneration of the wound.

[0096] Step S7: In vivo experiments verify the regulatory effects of Hst1@PNPs on macrophage polarization, inflammatory factors, and oxidative stress factor expression.

[0097] In order to evaluate the ROS scavenging and anti-inflammatory ability of Hst1@PNPs, the corresponding parameters were detected by immunofluorescence, WB and Elisa. The results showed that the number of M1 macrophages in the Hst1@PNPs and Hst1 groups was significantly lower than that in the control group on the 3rd and 7th days after surgery. Quantitative analysis by plus software showed that compared with the control group, the number of M1 macrophages in the Hst1@PNPs and Hst1 groups decreased by 2.81 and 2.01 times, respectively, on the third day after surgery, and decreased by 2.00 and 1.63 times, respectively, on the seventh day after surgery. In addition, the number of M2 macrophages in the Hst1@PNPs group was significantly higher than that in the control group on the third and seventh days after surgery. Quantitative analysis showed that compared with the control group, the number of M2 macrophages in the Hst1@PNPs and PNPs groups increased by 2.56 and 2.28 times, respectively, on the third day after surgery, and increased by 2.00 and 1.88 times, respectively, on the seventh day after surgery. The expression of M1 macrophage marker iNOS and M2 marker CD206 was further quantified by WB. On the third day after surgery, the expression of iNOS in the Hst1@PNPs, Hst1 and PNPs groups was significantly lower than that in the control group, and the wounds treated with Hst1@PNPs significantly promoted the expression of CD206.

[0098] Subsequently, Elisa test was used to evaluate the effect of Hst1@PNPs on the expression levels of proinflammatory factors such as IL-1β, IL-6 and TNF-α, pro-wound healing factors such as IL-10 and oxidative stress products such as ROS, MDA and SOD2. On the 7th day after surgery, the expression levels of proinflammatory factors in the Hst1@PNPs and Hst1 groups were significantly lower than those in the control group. On the 3rd day after surgery, the expression of IL-10 in the Hst1@PNPs group was significantly higher than that in the control group, and on the 7th day after surgery, it was significantly lower than that in the control group. On the 3rd and 7th days after surgery, the expression levels of ROS and MDA in the Hst1@PNPs and Hst1 groups were significantly lower than those in the control group. On the 7th day after surgery, the expression level of SOD2 in the Hst1@PNPs and Hst1 groups was significantly higher than that in the control group. In summary, Hst1@PNPs can accelerate the healing of diabetic wounds by improving the adverse microenvironment of persistent inflammation and excessive ROS in diabetic wounds.

[0099] Step S8: High-throughput sequencing to detect the regulatory effect of Hst1 on RAW264.7 macrophages.

[0100] To explore the mechanism of Hst1 in diabetic wound healing, high-throughput sequencing was performed to identify the enriched signaling pathways and biological processes of target genes. Compared with the control group, 1861 genes were downregulated and 2827 genes were upregulated in RAW264.7 macrophages treated with 100 ng / mL LPS. GO analysis showed that these differential genes in the LPS group were enriched in innate immune response, inflammatory response, cellular response to LPS, and positive regulation of TNF and IL-1β production, indicating that LPS successfully induced the polarization of M1 macrophages. Similarly, KEGG analysis showed that most of the differential genes in the LPS group were enriched in chemokine signaling pathways, cytokine-cytokine receptor interactions, cancer pathways, and phagosomes.

[0101] Compared with the LPS group, 202 genes were found to be down-regulated and 402 genes were found to be up-regulated in RAW264.7 macrophages treated with LPS+Hst1. GO analysis showed that the differentially expressed genes related to wound repair in the LPS+Hst1 group were mainly related to the structural components of the extracellular matrix, laminin and glycosaminoglycan binding, and collagen-containing extracellular matrix. ECM components such as fibronectin, collagen, laminin and proteoglycans provide a microenvironment conducive to cell recruitment and differentiation, thereby promoting tissue repair and regeneration. In addition, KEGG pathway analysis showed that most of the differentially expressed genes in LPS+Hst1 were significantly involved in metabolic pathways, cytokine-cytokine receptor interactions and PI3K-Akt signaling pathways. These findings indicate that the PI3K-AKT signaling pathway is significantly activated, which is essential for cell growth, movement, metabolism and development, and plays a key role in promoting wound healing. Overall, Hst1 may synergistically promote diabetic wound healing by regulating the respective signaling pathways involved in wound repair.

[0102] This article uses specific examples to illustrate the principles and implementation methods of the present invention. The above examples are only used to help understand the method and core ideas of the present invention. The above is only a preferred implementation of the present invention. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of the present invention, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the inventive concept and technical solution to other occasions without improvement, should be regarded as the protection scope of the present invention.

Claims

1. A controlled release method for promoting healing of diabetic wounds, characterized in that: The treatment experiment steps include the following: Step S1: Preparation of Hst1@PNPs, 50 ml of whole blood was drawn from the volunteers. The collected blood was centrifuged for the first time at 600 g for 10 minutes to remove the red blood cells at the bottom layer. The second centrifugation was performed at 3000 rpm for 20 minutes to remove the upper serum layer. The platelet precipitate was washed with 1 ml of The platelets were suspended in PBS to obtain a platelet concentration of 1249×10^9 / L. The concentrated platelets were repeatedly frozen and thawed three times at -80℃ and 25℃, and then centrifuged at 12000g for 10 minutes at 4℃ to collect the platelet membrane (PM) precipitate. The platelet membrane was fully ground with a grinding rod and then centrifuged at 3200g for 5 minutes. The precipitate was suspended in PBS, and the suspension was extracted with a syringe and installed in an engineered vesicle extrusion device. It was repeatedly passed through a 0.8um membrane and a 0.2um membrane for 20 times to obtain PNPs and stored at -80℃. The concentrations of PM and PNPs were detected using a BCA kit (P0012S, Shanghai Biyuntian Biotechnology Co., Ltd., Shanghai, China), which were 7.16g / L and 4.37g / L, respectively. PNPs were mixed with Hst1 and shaken at 4℃ for 12 hours to obtain Hst1@PNPs. Step S2: Coomassie Brilliant Blue staining, 10% separation gel and stacking gel were prepared. After the gel was formed, the glass plate was placed in the electrophoresis tank, 1× electrophoresis solution was added, protein markers, PM, and PNPs were added to the sample wells, and the gel was run at 80 V for 20 min, then at 120 V for 60 min. The gel was cut and soaked in distilled water for 5 min, and then Coomassie Brilliant Blue staining solution (G1042, Beijing Labread Trading Co., Ltd., Beijing, China) was added, incubated on a shaker for 1 h, destained with tap water for 30 min, and then photographed with a chemiluminescence imaging system; Step S3: Characterization of Hst1@PNPs, The Hst1, PNPs, and Hst1@PNPs samples were dehydrated and frozen under liquid nitrogen, then cut into thin slices with a diamond blade, a layer of conductive material was coated on the samples using a vacuum sputtering instrument, and then placed in a SEM (Nova Nano 450, Thermo Fisher Scientific, USA) and TEM (Talos F200, Thermo Fisher Scientific, USA) chamber, and the surface and internal structure and morphology of the samples were detected by scanning and transmission electron microscopy. The PNPs, Hst1, and Hst1@PNPs samples were diluted with ultrapure water to a particle concentration suitable for dynamic light scattering (DLS) analysis, and the particle size of these samples was detected using an Anton Paar Litesizer 100 device and a fluorescence spectrometer (NanoDrop3300, Thermo Fisher Scientific, USA); Step S4: In vitro sustained release of Hst1@PNPs, 1 ml of Hst1@PNPs was added to a dialysis bag with a pore size of 8000 kDa, which was then placed in a 15 ml EP tube filled with 5 ml of distilled water. After 0.5 hours, 1.5 hours, 3 hours, 6 hours, 8 hours, 12 hours and 24 hours, 100 ul of solution was taken from the 5 ml of distilled water, and these samples were added to a 96-well plate, and the OD value was detected at a wavelength of 450 nm using an enzyme reader. The exudate from the diabetic wound of the rat was collected, and then the collected exudate was mixed with Hst1 and Hst1@PNPs in a volume ratio of 1:1, and 1 ml of the mixture was added to the dialysis bag. The subsequent steps were the same as above. The OD values ​​of these samples were detected after 0.5 hours, 1 hour, 3 hours, 6 hours, 8 hours, 12 hours and 24 hours, and then graphed using Graphpad prism9; Step S5: In vitro adsorption of LPS, 10 ng / ml LPS was mixed with 10 ug / ml, 100 ug / ml, and 500 ug / ml of PNPs, respectively, and then fully shaken on a shaker at 37 °C for 1 h. The mixture was centrifuged at 4500 rpm for 6 min, and the supernatant was collected. The transmittance of the supernatant was measured using a Fourier transform infrared spectrometer (Nicolet iS50 FTIR, Thermo Fisher Scientific, USA), and the LPS concentration in the supernatant was measured using a horseshoe crab kit (EC32545, Xiamen Boendu Biotechnology Co., Ltd., Xiamen, China) according to the instructions; Step S6: Cell culture, RAW 264.7 macrophages, HaCaT cells, and human skin fibroblasts were cultured in DMEM supplemented with 10% fetal bovine serum and 1% antibiotics, and the cells were cultured in an incubator at 37°C and 5% CO2; Step S7: CCK-8 cytotoxicity assay, RAW 264.7 cells were seeded in 96-well plates at a density of 2×10^4 cells per well and incubated overnight. In previous studies, 10 μM Hst1 was verified to be the optimal concentration for promoting skin cell migration, adhesion and acute wound healing. Therefore, 10 μM Hst1 was used as a positive control in this study. The cells were treated with 10 μM Hst1, 0.1 μg / ml, 0.5 μg / ml, 1 μg / ml PNPs, Hst1@0.1 μg / ml PNPs, Hst1@0.5 μg / ml PNPs, and Hst1@1 μg / ml PNPs for 48 hours, respectively. At 24 hours and 48 hours after treatment, 10 μl of CCK-8 solution was added to each well and incubated for another hour. Subsequently, the absorbance at a wavelength of 450 nm was measured using a microplate reader. According to the experimental results, the optimal concentration of 1 μg / ml PNPs was used in subsequent experiments. Step S8: Cell scratch test, HaCaT cells and human skin fibroblasts were seeded into 6-well plates until 100% confluence was achieved. Two straight lines were drawn in each well using a sterile 200 μl pipette tip. After rinsing with PBS, the cells were exposed to the control group (DMEM+1% FBS), the experimental groups 10 μM Hst1+DMEM+1% FBS, 1 μg / ml PNPs+DMEM+1% FBS and Hst1@1 μg / ml PNPs+DMEM+1% FBS, respectively. Immediate images after scratching, as well as images at 24 h, 48 h, 72 h and 96 h after scratching were taken using a Nikon optical microscope. The unclosed area of ​​the scratch wound was measured using Image J software, and the percentage of wound healing was calculated using the formula: percentage of wound healing (%) = [(A0-Ax) / A0] × 100%, where A0 represents the initial wound area, and Ax represents the wound area at 24 h, 48 h, 72 h and 96 h; Step S9: DCFH-DA experiment, RAW264.7 macrophages were seeded in 24-well plates at a density of 1×10^5 cells per well and grown to 60%. The cells were treated with Hst1, PNPs, and Hst1@PNPs for 8 h, respectively. Then, the medium was removed, and 100 ng / ml of LPS was added to each well (except the control group) to induce ROS generation and incubated for 24 h. Subsequently, 100 μl of serum-free medium containing 0.1 μl DCFH-DA (S0033S, Shanghai Bio-Technology Co., Ltd., Shanghai, CN) was added to each well and incubated at 37 °C in the dark for 50 min. 0.1 μl of Hochest was added to each well to stain the cell nucleus for 10 min, and then rinsed with PBS three times and photographed with a fluorescence confocal microscope (Nikon Eclipse TI-SR, Tokyo, Japan); Step S10: In vitro immunofluorescence assay, RAW264.7 cells were seeded in 24-well plates at a density of 1×10^5 cells per well and incubated overnight. Subsequently, the cells were treated with control, LPS, LPS+Hst1, LPS+PNPs, and LPS+Hst1@PNPs for 24 hours, respectively. After rinsing with PBS three times, the cells were fixed in 4% paraformaldehyde for 20 minutes, rinsed with PBS again for three times, and 0.5% Triton-X-100 was added to the plate. After incubation at room temperature for 10 minutes, the cells were blocked with 5% bovine serum albumin (BSA) for 30 minutes, and then the primary antibodies iNOS (53-5920-80; 1:100; Thermo Fisher Scientific Co., Ltd, California, US) and CD206 (ab300621; 1:50; Abcam Trade Co., Ltd, Shanghai, CN) at 37°C in the dark for 2 h, and then incubated with the corresponding fluorescent secondary antibody for another 1 h at room temperature. The cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 10 min and photographed using a fluorescent confocal microscope. In order to distinguish between M1 and M2 macrophages, iNOS positive staining was used to characterize M1 macrophages, and CD206 positive staining was used to characterize M2 macrophages; Step S11: In vitro ELISA, After the cells were treated under different conditions, the culture supernatant of each well was collected and ELISA kits were used to measure IL-6 (RE3262-96T; Bioroyee Biotech Co., Ltd, Beijing, CN), IL-1β (WKQ-SJH-01413-96T; Wekeqi Biotech Co., Ltd, Chengdu, CN), and TNF-α (1217202; Dakewei Biotechnology Co., Ltd., Shenzhen, CN), and the absorbance was measured at the corresponding wavelength using a microplate reader, and the concentrations of these cytokines were calculated using the standard curve formula; Step S12: high-throughput sequencing, RAW264.7 macrophages were seeded in 12-well plates and then exposed to the control group, LPS group, and LPS+Hst1 group for 24 hours to induce macrophage polarization. Then, total RNA was extracted using Trizol reagent, 1 μg of total RNA was used for subsequent library preparation, poly(A) mRNA was isolated using Oligo(dT) beads, mRNA fragmentation was performed using divalent cations and high temperature, primer synthesis was performed using random primers, first-strand cDNA and second-strand cDNA were synthesized, and the purified double-stranded cDNA was subjected to end repair and A-tailing reaction, and adapters were added to both ends by TA ligation. Then, P5 and P7 primers were used to perform PCR amplification for each sample, and the PCR products were verified. Next, the libraries with different indexes were multiplexed and loaded onto the Illumina HiSeq / Illumina Novaseq / MGI2000 instruments, and 2x150 paired-end (PE) configuration sequencing was performed according to the instructions. The final library was quantified by a fluorometer and analyzed using Bioanalyzer The 2100 system checked the size distribution to ensure that it met the specific requirements of the sequencing platform, and then GO and KEGG analyses were performed on the differentially expressed genes; Step S13: Animal experiment, Thirty male C57 mice aged 6–8 weeks and weighing 22–28 g were purchased from Guangdong Animal Research Center, China. All animal experiments were approved by the Animal Ethics Committee of the Dermatology Hospital of Southern Medical University. Before the experiments, the mice were allowed to adapt to the living environment for one week; Step S14: Diabetic mouse wound healing experiment, Thirty C57 mice were prepared and fed with a standard diet for 3 days, then changed to a 60% high-fat diet for one week. The mice were fasted overnight, then marked with ear tags and fasting blood glucose levels were measured. Subsequently, a 10 mg / mL STZ solution was prepared using pH 4.5 sodium citrate, and STZ was then injected intraperitoneally at 100 mg / kg. STZ injections were performed on days 1, 3, and 5, respectively. Blood glucose levels were monitored every day after the three injections. If blood glucose returned to normal levels, additional injections were performed. Once blood glucose levels were stable for a week (>16.7 mmol / L), the mice could be selected for the diabetic wound model. Finally, 18 mice were successfully modeled. The treatments were divided into four groups: control group (0.9% NaCl), Hst1 group, PNPs group and Hst1@PNPs group. The mice were anesthetized by intraperitoneal injection of 1% sodium pentobarbital (5 ml / kg). The hair on the back of each mouse was carefully shaved and disinfected with iodine. Four acute full-thickness skin defect models were made in the back area of ​​each mouse using a 0.6 cm diameter biopsy device. Then, silicone rings were glued to the wound surface to prevent wound contracture. The four treatments were randomly applied to four wounds of each mouse for a self-control study. Then, 0.5 ml of the drug was given to each wound every other day until the wound was completely healed. The wounds of the mice were photographed on days 0, 3, 5, 7, 10 and 14 after surgery, and then the images were taken using ImageJ. The wound area of ​​mice was measured by J software, and the wound healing percentage was calculated, wound healing percentage (%) = [(A0-Ax) / A0] × 100%, A0 represents the initial wound area on day 0, Ax represents the wound area on days 3, 5, 7, 10 and 14, 5 mice were sacrificed at each time point on days 3, 7 and 14 after surgery, and the wound specimens were completely removed. The samples were divided into two parts, one part was fixed with 4% paraformaldehyde solution for subsequent histological staining, and the other part was used for Western blotting and ELISA detection; Step S15: Histological staining, Hematoxylin-eosin staining; Masson's trichrome staining: tissue sections were baked at 70°C for 1 hour, then dewaxed and rinsed with running water for 2 minutes, stained with iron hematoxylin for 4 minutes, then rinsed with running water, differentiated with 1% hydrochloric acid alcohol for 1 second, then rinsed with running water for 2 minutes to make it blue, tissue sections were stained with picric acid for 3 minutes, phosphomolybdic acid for 4 minutes, and aniline blue for 1 minute, then quickly rinsed and baked in an oven for 30 minutes, images were taken using an electron microscope and analyzed using Image-ProPlus software, 5 tissue sections were selected from each group for photography, and the expression level of new collagen was quantified using the following formula: average optical density value (AOD) = total optical density (IOD) value / area sum; in vivo immunofluorescence staining: sections were stained with ethylenediaminetetramine at pH = 9.0 The sections were fixed with EDTA solution for 15 min and blocked with 5% bovine serum albumin (BSA) at 37°C for 30 min. The sections were incubated with primary antibodies CD31 (AF3628, 1:200, Bio-Techne, Minneapolis, USA), iNOS (53-5920-80, 1:100, Thermo Fisher Scientific, California, USA), and CD206 (ab300621, 1:50, Abcam Trading Co., Ltd., Shanghai, China) at 4°C overnight in the dark. The corresponding fluorescent secondary antibodies were added and incubated at room temperature for 1 h. The sections were then blocked with 4-6-diamidino-2-phenylindole (DAPI) anti-fluorescence quencher. Five tissue sections in each group were photographed using a fluorescence confocal microscope and Image-Pro Plus software was used to measure the fluorescence area of ​​CD31, and the number of M1 and M2 macrophages was automatically counted and analyzed using Image-Pro Plus software; Western blot, in order to quantify the expression of M1 and M2 macrophages, wound tissue samples were taken for Western blot detection. First, the tissue was homogenized in lysis buffer using a homogenizer, and then the homogenate was centrifuged at 12000g for 10 minutes to obtain the supernatant. The protein concentration was determined using a BCA kit, and the sample was mixed with the loading buffer, boiled and added to the SDS-PAGE gel wells. 80 volts were selected for electrophoresis for 30 minutes, and then switched to 120 volts for electrophoresis for 1 hour. The gel was then transferred to a PVDF membrane, the membrane was blocked with skim milk powder, and then incubated with primary antibodies iNOS (ab283655, 1:1000, Abcam Trading Co., Ltd., Shanghai, China) and CD206 (ab64693, 1:1000, Abcam Trading Co., Ltd., Shanghai, China) at 4°C overnight. Subsequently, the secondary antibody was incubated at room temperature for 1 hour and controlled release was performed using chemiluminescence. Image J software to analyze the Western blot bands to detect the expression levels; Step S16: in vivo ELISA, To quantify the expression of inflammatory and oxidative stress factors, wound tissue samples were homogenized in lysis buffer using a homogenizer and then centrifuged at 12,000 g for 10 min to obtain the supernatant, and the levels of IL-6, IL-1β, IL-10, TNF-α, ROS (JL20383; Shanghai Jianglai Biotechnology Co., Ltd., Shanghai, China), malondialdehyde (MDA) (MM-0897M1; Guangzhou Anfei Biotechnology Co., Ltd., Guangzhou, China), and superoxide dismutase 2 (SOD2) (MM-0389M1; Guangzhou Anfei Biotechnology Co., Ltd., Guangzhou, China) were measured using ELISA kits, and the subsequent steps were the same as the above cell ELISA experiments; Step S17: Statistical analysis, Statistical analysis was performed using one-way ANOVA or two-way ANOVA followed by Bonferroni test. All numerical results were expressed as mean ± standard deviation (SD). A significance level of P < 0.05 was considered statistically significant.

2. The controlled release diabetic wound healing treatment method according to claim 1, characterized in that: The treatment experiment effect verification steps include the following: Step S1: Hst1@PNPs characterization, First, PNPs were synthesized and Coomassie Brilliant Blue staining confirmed that PNPs retained most of the protein distribution in PM. Electron microscopy revealed that Hst1 presented a square structure and could be encapsulated by PNPs with a ring-shaped lipid membrane structure to form a nanoparticle structure. DLS analysis showed that the particle sizes of PNPs, Hst1, and Hst1@PNPs were approximately 174.1 nm, 636.1 nm, and 1216.0 nm, respectively. The particle size of PNPs increased by hundreds of times after encapsulating Hst1, indicating that Hst1 was not only encapsulated in PNPs, but also likely bound to the surface membrane proteins of PNPs. Studies have shown that Hst1 is degraded to 46% and 8% after incubation with chronic wound exudate for 8 hours and 24 hours, respectively. The half-life of platelets in systemic circulation is about 30 hours, which is partly attributed to the interaction between CD47 on them and signal regulatory protein α (SIRPα) on immune cells to prevent them from being cleared by the immune system. PNPs retain these characteristics of platelets and can achieve sustained release and targeted delivery of drugs when encapsulated. In this study, we encapsulated Hst1 in PNPs to achieve sustained release, and verified it through in vitro release experiments. Through dialysis, Hst1 can cross to the external solution through a dialysis bag with a certain pore size, and then collect the solution to detect OD The results showed that Hst1@PNPs can release Hst1 slowly over time and reach a plateau at 8 hours. Subsequently, in the diabetic wound exudate of rats, Hst1 was degraded to 33.6% and 50.4% of its original amount after 0.5 hours and 24 hours of co-incubation, respectively. Obviously, Hst1@PNPs was more stable, with 90.1% and 76.1% remaining after 0.5 hours and 24 hours. Therefore, compared with the previous study that only 8% of Hst1 was degraded after 24 hours, Hst1@PNPs can increase the Hst1 retention rate by 9.5 times in the high protease environment of diabetic wound exudate, greatly improving the treatment efficiency and reducing the frequency of administration. LPS can promote macrophage polarization to M1 phenotype, which can initiate pro-inflammatory response and secrete pro-inflammatory factors such as IL-6, IL-1β and TNF-α. In this study, three different concentrations of PNPs (10ug / ml, 100ug / ml, 500ug / ml) were used to adsorb 10ng / mL LPS, and the transmittance of the solution was detected by infrared spectrometer. It was found that the transmittance of PNPs with a concentration of 100ug / ml was much higher than that of PNPs with other concentrations, indicating that the adsorption effect of LPS was more obvious. In addition, the use of chromogenic matrix horseshoe crab kit confirmed that PNPs with three concentrations can significantly adsorb LPS. Therefore, PNPs can adsorb excess LPS in diabetic wound exudate to improve the microenvironment of persistent inflammation and high-intensity oxidative stress, and synergize with Hst1 to promote wound healing. Previous studies have confirmed that Hst1 can promote the adhesion, crawling and migration of various cells. In the concentration range of 0.5uM to 400uM, LDH experiments confirmed that no obvious cytotoxicity was found at any concentration. In addition, PNPs are derived from autologous platelets, have high biosafety and low immunogenicity, and no toxic reagents are added during the preparation process. In this study, the CCK8 kit was used to detect the cytotoxicity of three different concentrations of Hst1@PNPs, and it was found that there was no obvious cytotoxic effect on R264.7 macrophages after 24 hours and 48 hours of treatment; Step S2: In vitro experiments to verify the effect of Hst1@PNPs on the migration of HaCaT cells and fibroblasts. In order to study the effect of Hst1@PNPs on skin cell migration, scratch tests were performed on HaCaT cells and human skin fibroblasts. The experiments were divided into control group, 10μM Hst1, PNPs and Hst1@PNPs groups. The results showed that compared with other groups, the scratch width of HaCaT cells and fibroblasts treated with Hst1@PNPs was significantly reduced. Similarly, the wound area of ​​HaCaT cells treated with Hst1@PNPs was significantly reduced, and more than 80% healed after 48 hours. The control group and 10μM Hst1 positive control group had no significant promoting effect on the migration of HaCaT cells and fibroblasts (P<0.05, Figure 3D-E). The above results show that Hst1 and PNPs have a synergistic effect on cell migration. Step S3: In vitro experiments to verify the ability of Hst1@PNPs to scavenge ROS. To verify the effect of Hst1@PNPs on ROS scavenging, the ROS level was evaluated by immunofluorescence. The results showed that the intracellular ROS production of Raw264.7 macrophages induced by LPS increased significantly in the LPS group, while the ROS level was significantly reduced in the LPS+Hst1, LPS+PNPs, and LPS+Hst1@PNPs groups and restored the ROS level to the level of the control group. Therefore, Hst1 and PNPs can not only clear LPS-induced ROS production alone, but also achieve a synergistic effect in ROS scavenging. Step S4: In vitro experiments verify the effect of Hst1@PNPs on macrophage polarization and expression of pro-inflammatory factors in vitro. In order to study the effect of Hst1@PNPs on macrophage polarization, the expression levels of M1 and M2 macrophages were detected by immunofluorescence. Compared with the control group, LPS significantly increased the expression of iNOS, a marker of M1 macrophages, and successfully induced polarization to M1 phenotype. Compared with LPS and control groups, Hst1, PNPs and Hst1@PNPs groups significantly reduced the expression of iNOS and increased the expression of CD206, a marker of M2 macrophages. Therefore, Hst1@PNPs can promote the transformation of M1 to M2 macrophages and M0 to M2, thereby promoting the transition of diabetic wound healing process from inflammatory phase to granulation phase and remodeling phase. In addition, to evaluate the effect of Hst1@PNPs on the production of inflammatory factors in LPS-induced macrophages, the expression levels of IL-1β, IL-6, and TNF-α were detected using Elisa kits. Compared with the control group, LPS significantly increased the expression of pro-inflammatory factors, while Hst1@PNPs could significantly reduce the expression of IL-1β and TNF-α compared with the LPS group. In addition, the IL-6 levels in the PNPs and Hst1@PNPs groups were significantly higher than those in the LPS and control groups. Step S5: Verify the sustained release effect of Hst1@PNPs in vivo and its effect on promoting wound healing in diabetic mice skin. A diabetic wound model was constructed to evaluate the sustained-release effect of Hst1@PNPs on diabetic wounds. Mice with blood glucose levels exceeding 16.7mmol / L for one week were identified as being useful for diabetic wound model construction. On the 3rd, 5th, 7th, 10th, and 14th days after surgery, the healing rate of diabetic wounds treated with Hst1@PNPs was faster than that of other groups. Quantitative analysis further confirmed that Hst1@PNPs significantly promoted diabetic wound healing on the 3rd and 5th days after surgery, with wound healing percentages reaching 6.85 times and 2.13 times that of the control group on the 3rd and 5th days after surgery, respectively. However, there was no statistical difference between the Hst1 group and the PNPs group and the control group. These results indicate that when Hst1 is applied to diabetic wounds, it is rapidly degraded by high proteases, while Hst1@PNPs significantly promotes diabetic wound healing through its sustained-release effect. Step S6: In vivo experiments verify the effects of Hst1@PNPs on wound re-epithelialization, collagen expression and angiogenesis. In this study, the effects of Hst1@PNPs on epithelial migration, collagen deposition, and angiogenesis were evaluated by pathological tissue staining. As time went on, the epidermis in the wound gradually migrated toward the center of the wound until it closed. Quantitative analysis showed that on the 3rd and 7th days after surgery, the length of the unclosed wound in the control group was significantly longer than that in the other groups. Hst1@PNPs showed the fastest epidermal migration speed among the four groups. On the 7th and 14th days after surgery, the collagen expression level in the Hst1@PNPs group was significantly higher than that in the control group. In order to study the effect of Hst1@PNPs on angiogenesis, the expression of CD31-positive blood vessels was detected by immunofluorescence staining. On the 7th day after surgery, the CD31-positive blood vessel fluorescence area in the Hst1@PNPs group and the PNPs group was significantly higher than that in the control group. However, there was no statistical difference in the Hst1 group in promoting the expression of CD31-positive blood vessels. On the 14th day after surgery, there was no statistical difference between the groups. Overall, these results showed that compared with Hst1 alone, Hst1@PNPs significantly promoted the structural integrity and functional regeneration of the wound. Step S7: In vivo experiments verify the regulatory effects of Hst1@PNPs on macrophage polarization, inflammatory factors, and oxidative stress factor expression. In order to evaluate the ROS scavenging and anti-inflammatory ability of Hst1@PNPs, the corresponding parameters were detected by immunofluorescence, WB and Elisa. The results showed that the number of M1 macrophages in the Hst1@PNPs and Hst1 groups was significantly lower than that in the control group on the 3rd and 7th days after surgery. Quantitative analysis by plus software showed that compared with the control group, the number of M1 macrophages in the Hst1@PNPs and Hst1 groups decreased by 2.81 and 2.01 times, respectively, on the third day after surgery, and decreased by 2.00 and 1.63 times, respectively, on the seventh day after surgery. In addition, the number of M2 macrophages in the Hst1@PNPs group was significantly higher than that in the control group on the third and seventh days after surgery. Quantitative analysis showed that compared with the control group, the number of M2 macrophages in the Hst1@PNPs and PNPs groups increased by 2.56 and 2.28 times, respectively, on the third day after surgery, and increased by 2.00 and 1.88 times, respectively, on the seventh day after surgery. The expression of M1 macrophage marker iNOS and M2 marker CD206 was further quantified by WB. On the third day after surgery, the expression of iNOS in the Hst1@PNPs, Hst1 and PNPs groups was significantly lower than that in the control group, and the wounds treated with Hst1@PNPs significantly promoted the expression of CD206. Subsequently, Elisa test was used to evaluate the effect of Hst1@PNPs on the expression levels of proinflammatory factors such as IL-1β, IL-6 and TNF-α, pro-wound healing factors such as IL-10 and oxidative stress products such as ROS, MDA and SOD2. On the 7th day after surgery, the expression levels of proinflammatory factors in the Hst1@PNPs and Hst1 groups were significantly lower than those in the control group. On the 3rd day after surgery, the expression of IL-10 in the Hst1@PNPs group was significantly higher than that in the control group, and on the 7th day after surgery, it was significantly lower than that in the control group. On the 3rd and 7th days after surgery, the expression levels of ROS and MDA in the Hst1@PNPs and Hst1 groups were significantly lower than those in the control group. On the 7th day after surgery, the expression level of SOD2 in the Hst1@PNPs and Hst1 groups was significantly higher than that in the control group. In summary, Hst1@PNPs can accelerate the healing of diabetic wounds by improving the adverse microenvironment of persistent inflammation and excessive ROS in diabetic wounds. Step S8: High-throughput sequencing to detect the regulatory effect of Hst1 on RAW264.7 macrophages. To explore the mechanism of Hst1 in diabetic wound healing, high-throughput sequencing was performed to identify the enriched signaling pathways and biological processes of target genes. Compared with the control group, 1861 genes were downregulated and 2827 genes were upregulated in RAW264.7 macrophages treated with 100 ng / mL LPS. GO analysis showed that these differential genes in the LPS group were enriched in innate immune response, inflammatory response, cellular response to LPS, and positive regulation of TNF and IL-1β production, indicating that LPS successfully induced the polarization of M1 macrophages. Similarly, KEGG analysis showed that most of the differential genes in the LPS group were enriched in chemokine signaling pathways, cytokine-cytokine receptor interactions, cancer pathways, and phagosomes. Compared with the LPS group, 202 genes were found to be down-regulated and 402 genes were found to be up-regulated in RAW264.7 macrophages treated with LPS+Hst1. GO analysis showed that the differentially expressed genes related to wound repair in the LPS+Hst1 group were mainly related to the structural components of the extracellular matrix, laminin and glycosaminoglycan binding, and collagen-containing extracellular matrix. ECM components such as fibronectin, collagen, laminin and proteoglycans provide a microenvironment conducive to cell recruitment and differentiation, thereby promoting tissue repair and regeneration. In addition, KEGG pathway analysis showed that most of the differentially expressed genes in LPS+Hst1 were significantly involved in metabolic pathways, cytokine-cytokine receptor interactions and PI3K-Akt signaling pathways. These findings indicate that the PI3K-AKT signaling pathway is significantly activated, which is essential for cell growth, movement, metabolism and development, and plays a key role in promoting wound healing. Overall, Hst1 may synergistically promote diabetic wound healing by regulating the respective signaling pathways involved in wound repair.

Images